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Method for culturing mammalian cells to improve recombinant protein production

a recombinant protein and cell culture technology, applied in the field of mammalian cell culture, can solve the problems of large-scale use of serum, inability to accurately detect and quantify inability to fully express the protein of interest, so as to improve cell viability, viable cell density, and protein expression. the effect of interes

Inactive Publication Date: 2010-09-02
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]The present invention provides a method comprising culturing an animal cell line expressing a protein of interest in serum free cell culture medium; the medium comprising spermine or spermidine at a concentration of at least about 0.10 μM, or putrescine at a concentration of at least about 100 μM. The medium may also comprise combinations of spermine, spermidine and putrescine at these concentrations. Cell viability, viable cell density and expression of the protein of interest are improved relative to cells grown in culture without spermine or spermidine at a concentration of at least about 0.10 μM, or putrescine at a concentration of at least about 100 μM. In some embodiments are provided mammalian cell lines, other embodiments provide Chinese Hamster Ovary (CHO) cell lines. Also provided are serum-free media that are also peptone-free.

Problems solved by technology

However, use of serum is problematic for large scale clinical manufacture of protein therapeutics.
Not only is large scale use of serum prohibitively expensive, serum is inherently uncharacterized, comprised of complex, unknown and unquantified components.
This inherent variability makes predictable large scale cell culture and / or recombinant protein production difficult and expensive.
In addition, removing serum proteins from downstream processing is burdensome.
Even without these difficulties, use of serum in a clinical manufacturing setting is highly undesirable from a regulatory point of view because animal serum brings with it the risk of contamination by viruses, mycoplasma and / or prions.
However, these hydrosylates are essentially undefined and, like animal sera, contain many complex, unknown and unquantified components.
Clinical manufacture of therapeutic proteins is an expensive, large scale endeavor.

Method used

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  • Method for culturing mammalian cells to improve recombinant protein production
  • Method for culturing mammalian cells to improve recombinant protein production
  • Method for culturing mammalian cells to improve recombinant protein production

Examples

Experimental program
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example 1

[0059]To test the effect of high concentration polyamine media formulations on cell culture performance a CHO cell line producing a recombinant IgG2 monoclonal antibody was seeded at 2.5×105 cells / ml in serum-free DMEM / F12 (SAFC, Lenexa, Kans.). DMEM / F12 contains 503 nM putrescine as a component of the commercial formulation. The media was supplemented with an additional 100-1000 μM putrescine dihydrochloride (Sigma-Aldrich, St. Louis, Mo.); 10-50 μM spermidine tetrachdrochloride (Sigma-Aldrich), or 10-50 μM spermine tetrahydrochloride (Sigma-Aldrich). Cells were maintained in suspension culture for three days at 36° C. in 5% CO2. Total cell density and viable cell density were measured using a Guava Easy-Cyte™ flow cytometer and Guava Viacount® Flex reagent (Hayward, Calif.) according to manufacture's instructions.

[0060]Following three days culture in the polyamine-supplemented-DMEM / F12 media, viable cell density and culture viability were measured, see FIGS. 1a and 1b. Mean viabil...

example 2

[0061]The antibody-expressing CHO cell line described in Example 1 was also grown in serum-free, soy hydrolysate containing media (VM-Soy, described in US Patent Application No. US2006-0115901), supplemented with spermine. VM-Soy basal media contains 0.98 μM (0.1620 mg / l) putrescine as a component of its formulation. The media was supplemented with spermine tetrahydrochloride (Sigma-Aldrich) to a concentration of 10-200 μM, each concentration was run in duplicate. Cells were seeded at 2.5×105 cells / ml and maintained for six days at 36° C. in 5% CO2. Viable cell density and cell viability were measured as described above. Once again cell viability and viable cell density were higher in those cultures supplemented with spermine compared to the unsupplemented control media (see FIGS. 2a and 2b). Viable cell density increased 8-23% and cell viability increased 22-29% compared to the unsupplemented control. The greatest increases in viable cell density and cell viability, 23% and 29% res...

example 3

[0064]To test the effect of spermine on cells in fed-batch production culture, four different recombinant monoclonal IgG2 antibody-producing CHO cell lines (each expressing a different IgG2 monoclonal antibody) were seeded at 5.0×105 cells / ml in serum-free, hydrolysate-free batch medium. The medium was an enriched formulation of DMEM / F12 medium (ingredients supplied by SAFC, Lenexa, Kans.). The medium contained putrescine at a concentration of 0.014 mM. In this experiment, cells were cultured in medium with or without 10 μM spermine tetrahydrochloride (Sigma-Aldrich). Cells were maintained in suspension culture for 11 days at 36° C., 5% CO2. Cultures were fed an enriched feed medium on the fourth, seventh, and ninth days of culture. The feed volume was equal to nine percent of the initial batch culture volume. The feed medium contained putrescine but not spermine and delivered 0.0025 mM putrescine as the final concentration in the cultures as a result of each feed. Glucose was also ...

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Abstract

The present invention relates to methods for mammalian cell culture, wherein the methods make use of media containing polyamines, such as putrescine, spermidine and spermine.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. provisional application Ser. No. 60 / 943,212, filed Jun. 11, 2007, the disclosure of which is relied upon and incorporated by reference herein.FIELD OF INVENTION[0002]The present invention relates to methods for mammalian cell culture, wherein the methods make use of media containing polyamines, such as putrescine, spermidine, and spermine.BACKGROUND OF INVENTION[0003]Many commercially important proteins are produced in mammalian cell lines that are adapted for long term growth in culture. Chinese hamster ovary (CHO) cell lines are well suited and widely used for recombinant production of therapeutic proteins, also known as “biologics”, including recombinant antibodies. CHO cell lines efficiently produce proteins that are correctly folded and have desired post-translational modifications. Further, CHO cell lines have gained acceptance and approval by regulatory agencies for use in clinical manufa...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/071
CPCC12N5/0031C12N2510/02C12N2500/46C12N5/0037C12N5/0043
Inventor MCCOY, REBECCA E.MORRIS, ARVIA E.
Owner AMGEN INC
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